The development of methods using capillary electrophoresis (CE) to measure and characterize synthetic nucleotide mixtures as well as intracellular nucleotide metabolites continues. Our previous work has shown that quantitative on-line sample-stacking produces a more than 100-fold signal enhancement for the CE analysis of nucleotides. These methods have been applied to characterize and study the hydrolytic and enzymatic stability of mixtures of synthetic nucleotide derivatives. Biologically derived mixtures of nucleotides, however, show a marked deterioration in signal and resolution upon sample-stacking and CE analysis because of the high ionic strength of these samples. Sample preparation methods and analysis strategies to overcome this effect have been explored and have provided some success, resulting in a 20- to 30-fold signal enhancement for biological samples. This investigation continues. We are also evaluating whole-column sample stacking for the micropreparative CE isolation of trace nucleotide metabolites for structural analysis by MALDI mass spectrometry. This will have application for the determination and measurement of the active intracellular nucleotide metabolites of the nucleoside-based reverse transcriptase inhibitors that are used in AIDS therapy. Nucleoside-based reverse transcriptase inhibitors comprise an important class of therapeutically useful drugs for AIDS. The Laboratory of Medicinal Chemistry has had a long-standing program studying conformationally locked nucleosides as potential anti-AIDS and anticancer agents. During the course of these studies it has come to be recognized that the conformation of nucleoside sugar can profoundly affect its chemical and biological properties. We have experimentally confirmed that this is indeed the case in two conformationally locked dideoxyadenosine (ddA) analogues where the orientation of the sugar oxygens lone pair orbitals are frozen to either enhance or impair the strength of the anomeric effect. Where the anomeric effect is enhanced, the glycosidic bond is lengthened and the nucleoside becomes more susceptible to hydrolysis. At pH 2, the conformationally locked ddA analogue with an enhanced anomeric effect has a depurination rate more than 6-fold greater than that of its structural isomer. Both ddA analogues, however, are more stable than ddA itself which has a half-life of only 2.67 min at pH 2.